Teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. F9 embryonal carcinoma cells are quite responsive to calcitonin. Exposure of F9 cells to retinoic acid induces differentiation to parietal endoderm cells. Differentiation to the endoderm cell type dramatically alters the hormonal responsiveness of the adenylate cyclase system; the cyclase of endodermal cells exhibits a low response to calcitonin while parathyroid hormone markedly enhances cyclic AMP formation. These variations in calcitonin and parathyroid hormone responsiveness suggest a possible regulatory role for these hormones during embryonic development. Conditions have been established for the growth of F9 cells under serum-free conditions in a medium supplemented with multiplication stimulating activity (MSA), transferrin, and fibronectin. At 100 ng/ml MSA completely replaces the growth requirement for fetal calf serum. Binding studies carried out with (I-125)MSA indicate about 55,000 MSA molecules bound per F9 cell with a Ka of 8.2 x 10 to the -9M. Insulin-like growth factors I and II both compete for binding, while insulin does not compete. These results indicate that MSA is capable of serving as an early embryonic growth factor.